Calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine brain has been purified by two methods, yielding enzymes with identical specific activities. SDS gel electrophoresis of the purified enzyme yields a single major polypeptide of 58,000 daltons. The catalytic properties of the enzyme in the presence of calmodulin, polyamines, and metal ions have been examined. The use of pH gradient chromatography on ion exchange gel and electrofocusing of the purified enzyme indicate (at least) two calmodulin-stimulated isoenzymes. Limited proteolysis of the purified enzyme yields a catalytically active peptide of approximately 40,000 daltons which does not interact with a calmodulin-Sepharose affinity gel.